Peptide fragments of myelin basic protein

ABSTRACT

Peptides containing regions of myelin basic protein found in the 86-102 and 143-168 sequences that are immunodominant in humans suffering from multiple sclerosis.

The United States Government has rights to this invention by virtue offunding from Grant Nos. NS 24247 and NS 29352 both from the NationalInstitutes of Health.

This is a continuation of application Ser. No. 08/046,354, filed Apr. 9,1993, now abandoned which is a continuation-in-part of (i) Ser. No.07/865,318, filed Apr. 9, 1992 now abandoned, which is acontinuation-in-part of Ser. No. 07/502,559, filed Mar. 30, 1990 nowabandoned; and (ii) Ser. No. 07/843,752, filed Feb. 28, 1992 nowabandoned.

FIELD OF THE INVENTION

This invention pertains to compositions and methods for suppression ofT-cell mediated or T-cell dependent autoimmune response. Morespecifically, the invention is directed to compositions comprisingpeptide fragments of myelin basic protein (MBP) or analogs thereof, andto methods of using such peptides and compositions to anergize, or tostop proliferation of, human T-cells specific for myelin basic protein,or to elicit active suppression of such T-cells. Peptides according tothe invention are also useful in identifying CD4⁺ T-cells reactive withmyelin basic protein.

BACKGROUND OF THE INVENTION

The discussion in this section is not limited to discussion of work thatqualifies as "prior art" against the present invention. Therefore, nosuch admission and no declaration against the present inventors'interests shall be implied by reason of this discussion.

Multiple Sclerosis (MS) is a chronic inflammatory disease of the whitematter of the human central nervous system and is believed to be ofautoimmune etiology. Regardless of its etiology, MS is accompanied byautoimmune attack of nerve tissue. For example, the disease ischaracterized by prominent T-cell and macrophage infiltrates intonervous tissue (i.e., the brain, spinal cord, peripheral nerves orassociated cell types), demyelination and neurological dysfunction.Myelin basic protein (MBP) has been extensively studied by the presentinventors, their co-workers and others as an autoantigen in the diseasebecause of its role as an inducing agent in the major animal model ofMS, experimental allergic encephalomyelitis (EAE), as well as its rolein the human disease post-viral encephalomyelitis. In addition, thepresent inventors and their co-workers have studied MBP as a "bystanderantigen" (Ser. No. 843,752, supra).

A major hypothesis regarding the pathogenesis of MS is that T-cellsreactive with myelin basic protein in the white matter of the CNSinitiate the inflammatory process. Another hypothesis is that T-cellsreactive with proteolipid protein (PLP) initiate the inflammatoryprocess. The demonstration that activated T-cells specific for myelinbasic protein (MBP) can be isolated from MS patients (Allegretta, M., etal., Science: 247: 778, 1990) further implicates MBP-reactive T-cells inthe pathogenesis of the disease. The work of the present inventors alsoshows that MBP-reactive T-cells are involved in the pathology of thedisease, subsequent to initiation of the inflammatory process. (As willbe described in more detail below, the present inventors demonstratedthat healthy individuals also often have MBP-specific T-cells, butunlike those of MS patients, MBP-specific T-cells from healthyindividuals are not activated.)

The current treatments for MS are solely palliative and involveadministration of drugs which act in a non-specific fashion to suppressthe immune response in the subject. Examples of such drugs arecyclophosphamide, Imuran (azathioprine) and Cyclosporin A. Steroidcompounds such as prednisone and methyl-prednisolone are also employedin many instances. These drugs have limited efficacy against MS. Use ofsuch drugs is limited by their toxicity and by the fact that they induce"global" immunosuppression upon. prolonged use, i.e., they alsodown-regulate the normal protective immune response to pathogenicmicroorganisms thereby increasing the risk of infection. In addition,patients that are globally immunosuppressed for prolonged periods oftime run an increased risk of developing certain malignancies.

More details on the immunological processes occurring are known forexperimental allergic encephalomyelitis (EAE), the primary animal modelfor MS. EAE can readily be induced in small mammals by immunization withmyelin basic protein (MBP) in an appropriate adjuvant or by adoptivetransfer through the injection of CD4⁺, MBP-reactive T-cells (Alvord Jr,E. C., et al. eds. in Experimental Allergic Encephalomyelitis: A UsefulModel for Multiple Sclerosis, A. R. Liss, N.Y., 1984; Makhtarian, D. E.,et al. Nature 305:356, 1984; Ben-Nun, A. et al. J. Immunol. 129:303,1982). The T-cells that induce EAE in both mice and rats, termedencephalitogenic cells, specifically recognize peptides corresponding tothe immunodominant regions of MBP. The presentation of these regions tothe T-cells occurs on the surface of antigen-presenting cells (APCs) inassociation with unique Major Histocompatibility Complex (MHC) class IImolecules. It should be emphasized that immunodominant regions of MBP,that is the portion of the protein most often recognized by MBP-reactiveT-cells of the CD4⁺ type, differs depending on the species of the hostmammal and may also differ depending on the species of MBP, despite thefact that the amino acid sequence of MBP exhibits very high interspecieshomology. For example, as the present inventors and their co-workershave discovered, an immunodominant epitope of human MBP in humans iscontained within the subsequence of the human MBP molecule comprisingamino acids 84-102. Another immunodominant epitope can be found in thesubsequence of the human MS molecule comprising amino acids 143-168.This is evidenced by the specificity of human T-cells isolated fromindividuals afflicted with MS (related patent application Ser. No.502,559 and Example 1 below). The immunodominant region of mouse MBP isamino acids 1-9 when administered to mice (Zamvil et al., Nature324:258, 1986) and that of rat MBP is amino acids 68-88 whenadministered to rats (Burns et al., J. Exp. Med. 169:27, 1989). On theother hand, the immunodominant region of guinea-pig MBP in rats islocated within residues 75-84 (Hashim, G. Myelin: Chemistry and Biology,Alan R. Liss, N.Y. 1980).

Based on the work done in the EAE system, alternative therapies arebeing developed for the treatment of autoimmune diseases in general andMS in particular. U.S. patent application Ser. No. 65,794 filed Jun. 24,1987 (now abandoned) and co-pending International Patent ApplicationPCT/US88/02139, filed Jun. 24, 1988, corresponding to national stageU.S. application Ser. No. 07/460,852, now abandoned, and acontinuation-in-part of this application, U.S. application Ser. No.07/596,936, now abandoned, disclose that oral or enteral administrationof whole myelin basic protein as well as disease-inducing andnon-inducing fragments and analogs thereof is effective in suppressingacute monophasic EAE and are useful in suppressing MS symptoms whensimilarly administered.

The following co-pending commonly assigned patent applications are alsoof interest: U.S. patent application Ser. No. 454,806 filed Dec. 20,1989, now abandoned, discloses the aerosol administration ofautoantigens, disease-suppressive fragments of said autoantigens andanalogs thereof as an effective treatment for treating T-cell mediatedautoimmune diseases such as MS.

U.S. application Ser. No. 07/487,732, filed Mar. 20, 1990, nowabandoned, entitled "Enhancement of the Down Regulation of AutoimmuneDiseases by Oral Administration of Autoantigens" discloses synergists(enhancers) for use with oral administration of autoantigens,disease-suppressive fragments and analogs thereof as effectivetreatments for T-cell mediated autoimmune diseases.

U.S. application Ser. No. 07/843,752, now abandoned, discloses methodsand compositions for treating autoimmune diseases orally or byinhalation by administering bystander antigens. Bystander antigens aretissue-specific antigens that are present at the locus of autoimmuneattack and that have the ability upon their being orally administered togenerate T-suppressor cells which in turn suppress immune attack at theafflicted tissue. Bystander antigens are not necessarily autoantigensand are not necessarily themselves the target of immune attack. (Infact, there is evidence that the immunosuppressive epitope(s) of anautoantigen are different from the immunodominant epitope(s) thereof,although immunodominant epitopes (which do elicit suppression upon oraladministration) may act as bystander antigens in suppressing immuneattack directed against other portions of the same antigen or portionsof other antigens in the afflicted tissue.) However, bystander antigensmust (a) be specific to the afflicted tissue and (b) possess the abilityto elicit T-suppressor cells upon oral administration.

It has now been found that oral administration of multiple doses andsmall amounts of whole antigens containing encephalitogenicimmunodominant epitopes elicits this type of active suppression. On theother hand, i.v. administration of entire autoantigen (or of one or moreencephalitogenic immuno-dominant epitope-containing fragments thereof)also induces suppression but only of immune attack T-cells recognizingepitopes of the autoantigen. The latter type of suppression, which isbelieved to proceed via the mechanism of clonal anergy, is also observedupon oral administration of single doses and large amounts of antigensencompassing encephalitogenic epitopes, specifically immunodominantepitopes, especially when such antigens are accompanied by proteaseinhibitors.

For human MBP, a protein believed to be an autoantigen for MS, extensivetesting by the present inventors has revealed fragments of the proteinincorporating epitopes which are recognized by a large number ofMBP-specific immune attack (CD4⁺) T-cells isolated from MS patients.Such fragments, comprising immunodominant epitopes, are likelycandidates for administration to patients suffering from MS, with thegoal of suppressing autoimmune response, and particularly suppressingthe function of MBP-reactive T-cells that are responsible for autoimmuneattack on nervous tissue. To that end, the present inventioncontemplates not only oral administration of such peptide fragments tomammals in need for such treatment but also parenteral administration ofsuch fragments.

Therefore, it is an object of the present invention to provideimmune-suppressive agents, specifically fragments of human MBP, andmethods of using these fragments to suppress human T-cell functions.

Another object of the present invention is to provide compositions andpharmaceutical formulations comprising these fragments of human MBPuseful for oral and/or i.v. administration to humans, and methods of useof such formulations.

Yet another object of this invention is to provide, as a reagent thatcould be used to determine the specificity of human T-cells, a peptidefragment of MBP incorporating an immunodominant epitope.

A further object is to provide compounds and compositions that anergizeMBP-reactive T-cells or cause active suppression of such T-cells, thelatter being evidenced for example by suppression of proliferation ofMBP-reactive T-cells.

These and other objects of the present invention will be apparent tothose of ordinary skill in the art in light of the presentspecification, drawings and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a bar graph showing the frequency of MBP reactive T-cellsisolated from MS patients (left panel, FIG. 1A) and healthy controls(right panel, FIG. 1B) that react specifically with different human MBPpeptide fragments.

FIG. 2 graphically illustrates the suppression of EAE (induced by i.p.inoculation with MBP-specific encephalitogenic T-cells) by transfer ofspleen cells from animals orally tolerized with MBP.

FIG. 3 depicts the suppression of adoptively transferred EAE byco-transfer of CD4⁺ -depleted or CD8⁺ -depleted T-cells from MBP fedanimals.

FIG. 4 is a bar graph of DTH responses associated with the extent ofprotection (it any) against EAE induction by co-transfer along with CD4⁺T-Cells of various T-cell subsets from MBP fed animals.

FIG. 5 is a bar graph of quantitative histologic analysis expressed inmean number of inflammatory foci isolated from the CNS (i.e., theparenchyma and the meninges) of mice injected with various T-cellsubsets from MBP-fed mice.

FIG. 6, in graph A, depicts the suppression of actively induced EAE byintravenous (IV) administration of MBP; FIG. 6, in graph B, depicts theeffect on adoptively transferred EAE of IV administration of MBP and theinability of spleen cells of IV-tolerized animals to confer suppressionwhen they are co-transferred to naive animals along with anencephalitogenic MBP line.

FIG. 7 is a bar graph showing the variation in suppression of EAEfollowing oral (7A) or IV (7B) administration of different MBP peptides.

FIG. 8 is a graphic representation of the ability of various peptidesconstructed based on the immunodominant epitope region of human MBP(human MBP amino acid residues Nos. 84-102) to stimulate proliferationof human MBP-reactive T-cell clones. Panel A (FIG. 8A): effect ofomitting one or more N-terminal amino acids; Panel B (FIG. 8B): effectof omitting one or more C-terminal amino acids.

FIG. 9 is a graph of the ability of a 15-mer (human MBP amino acidresidue Nos. 85-99) to stimulate proliferation of four different humanT-cell clones (FIGS. 9A, 9B, 9C, 9D) compared to the ability of MBPpeptides 84-102 and 86-97 to stimulate such proliferation.

FIG. 10 is a chart showing the TCR/MHC contacts for the 85-99 and 88-104human MBP peptides and a proposed motif for this interaction.

FIG. 11 is a chart showing the induction of proliferation of T-cellclones to the human 85-99 MBP peptides of the present invention ascompared to the proliferation induced in these clones by the native MBPprotein.

SUMMARY OF THE INVENTION

In one aspect, this invention is directed to immunosuppressive agentscomprising peptides which are fragments of human MBP. Embodiments of theinvention include peptides and pharmaceutical compositions comprisingthese peptides.

This invention is also directed to methods using the foregoing peptidesto suppress immune response against myelin basic protein and tissuescontaining it, and/or to suppress T-cells that recognize animmunodominant epitope of human MBP. These methods involve oral and/orparenteral administration of one or more peptides according to theinvention, and result in suppression of the immune response against theprotein and tissues containing it.

Also included in the present invention are fragments of human MBPcomprising the amino acid sequence ENPVVHFFKNIVTPR.

In a further aspect, the present invention is directed to pharmaceuticalcompositions comprising one or more of said fragments of MBP.

Additional aspects of the present invention involve methods of use offragments of human MBP in the suppression of human T-cell function, andin the identification of CD4⁺ MBP-reactive human T-cells.

DETAILED DESCRIPTION OF THE INVENTION

All patent applications, patents and publications cited in thisspecification are hereby incorporated by reference in their entirety. Inthe case of inconsistencies, the present disclosure, includingdefinitions, will prevail.

As used herein, "suppression" includes any measurable reproduciblereduction in T-cell proliferation in response to factors that normallystimulate those cells. However, it should be emphasized that the presentinvention is concerned only with the suppression of proliferation ofdeleterious T-cells, that is proliferation of T-cells that promoteautoimmune attack (CD4⁺ T-cells specific to a self-antigen, e.g. MBP).Indeed, an important aspect of the present invention is the ability toinduce suppression in a restricted manner, where the suppression ofdeleterious T-cells specific to a self antigen is the result of choiceof the fragment or fragments of MBP administered and/or the method ofadministration, as discussed below.

Suppression of the deleterious T-cell proliferation can also be measuredindirectly, i.e. as seen through a reduction in symptoms of a diseasewhich are directly or indirectly caused by immune attack T-cellproliferation, such as the damage to neural tissue observed in MS, orthe decrease in- the number or severity of attacks of MS suffered by MSpatients. Damage to neural tissue can be assessed for example bymagnetic resonance imaging (MRI) and measurement of the number andseverity of lesions visible therein. Reduction in MS attack number orseverity can be assessed for example by clinical evaluation of patients.Methods for both MRI and clinical evaluation are well-known in the art.

The term "autoantigen" is defined as any substance normally found withina mammal that, in an abnormal situation, is no longer recognized as"self" by the lymphocytes or antibodies of that mammal, and is attackedby the immunoregulatory system as though it were a foreign substance. Inother words, an autoantigen is an antigen that is subject to autoimmunedestruction. The mere presence of antibodies or even T-cells (of theCD4⁺ type) specific to a native antigen does not establish it as anautoantigen. MBP and PLP (proteolipid protein) are examples ofautoantigens in MS.

"Immunodominant epitope" of an autoantigen (such as MBP) means anantigenic determinant recognized by a substantial number including butnot limited to a majority (although not necessarily an absolutemajority) of T-cells of a sensitive mammal to which autoantigen suchT-cells will mount or help mount an immune response if the sensitivemammal is also an afflicted mammal. (It is evident from this discussionthat a "sensitive" mammal need not be an afflicted mammal.)

"Immunodominant regions" or "immunodominant domains" of an autoantigenare defined herein as those regions of the amino acid sequence of suchautoantigen containing an immunodominant epitope. The structures (and/orlocation within the MBP or other autoantigen molecule) of immunodominantepitopes (and regions) of MBP or other autoantigen vary depending on thehost and are, therefore, host-specific. The present inventors have infact adduced evidence that the reason the immunodominant epitopes arehost-specific is that they must comprise a motif (believed to becontained within a peptide fragment of about 8 to about 15 amino acidsin length) that binds to the major histocompatibility complex of thehost. This motif varies among different species (the MHC likewisevaries) and may also exhibit polymorphism among members of the samespecies.

The term "analog" of fragments of MBP includes compounds that are sostructurally related to the fragment of MBP that they possess the samebiological activity as the MBP fragment. The biological activityreferred to in this definition is the ability to suppress a T-cellmediated or T-cell dependent autoimmune response upon administration ofthe MBP fragment, or alternatively the ability to suppress proliferationof T-cells responsible for or contributing to autoimmune attack, or theability to be recognized by T-cells recognizing an immunodominantepitope of MBP. An example of an analog of the fragment MBP 84-102 isthe fragment MBP 84-102tyr, wherein amino acid 102 has been changed totyrosine. As can be seen from FIG. 8, this change has little or noeffect on the ability of the MBP fragment to stimulate proliferation ofthe MBP-reactive T-cell line. Furthermore, amino acid substitutions arenot expected to have any effect on solubility or pharmacokinetics of afragment because of the relatively small size of the present fragments.It should be noted that an "analog" need not display the same activityto the same degree, e.g., an "analog" does not need to be as potent asuppressor as an actual fragment of the native antigen.

Other analogs of the relevant human MBP epitopes could be constructedbased on ability of these analogs to bind the MHC and to be recognizedby the relevant T-cell receptor (both of which can be tested in vitro).

As used herein, "T-cells" or "T-lymphocytes" are defined as immunesystem cells, derived from stem cells located within hematopoietic (i.e.blood forming) tissues. There are three broad categories of T-cells:Helper, Suppressor and Cytotoxic. T-cells express either the CD4 antigen(and are then termed CD4⁺ T-cells) or the CD8 antigen (in which casethey are termed CD8⁺ T-cells) on their cell surface. The expression ofCD4 or CD8 antigens by peripheral (circulating) T-cells correlates withthe function and specificity of the T-cell. "Helper T-cells" which areCD4⁺ recognize antigens and Class II MHC molecules and perform helper orregulatory functions. "Cytotoxic" and "Suppressor" T-cells (which areCD8⁺) recognize antigens and Class I MHC molecules and perform cytotoxicand suppressor functions.

"Active suppression" is the suppression of immune function where thesuppression is the result of the induction of additional immune cells,specifically, regulatory (suppressor) T-cells.

"Clonal anergy" is the suppression of immune function by induction inimmune cells, specifically immune attack T-cells, of a state ofunresponsiveness and more particularly unresponsiveness to presentationof the antigen to which these cells are normally specific and to whichthey would normally proliferate. La Salle, J. et al, J. Exp. Med.,176:177-186, July 1992. Anergized T-cells appear normal in all respectsexcept they seem to be "turned off". They are not activated and--in theabsence of added interleukin-2 (IL-2)--they do not proliferate onpresentation of the antigen which they recognize. If IL-2 is added, thecells become de-anergized-and begin to proliferate on presentation ofantigen.

T-cells initiate an immune response when they encounterantigen-presenting cells (APCs), such as mononuclear phagocytes(macrophages, monocytes), Langerhan's cells or follicular dendriticcells, which initially take up, process (digest) and present antigenicfragments of a protein on their cell surface (in connection with theirMHC). CD4⁺ T-cells recognize antigen molecules exclusively when theprotein is processed, and peptide fragments thereof are presented, byAPCs that express Class II MHC molecules.

T-cell recognition of an antigen reflects a tri-molecular interactionbetween the T-cell receptor (TCR), the MHC molecule of the APC and apeptide or peptides processed by the APC via a cleft or pocket in thethree-dimensional structure of the Class II MHC molecule. (Bjorkman, P.J., et al., 1987, Nature, 329:506 and 329:512). The portion of theprotein most often presented on the APC surface, and recognized by theT-cell is the immunodominant epitope.

The present inventors have identified two regions of human MBP whichcontain immunodominant epitopes of human MBP in a human host. Theseepitopes are resident within two distinct portions of the human MBPamino acid sequence (residues Nos. 82-104 and 143-168 respectively). Asshown in Example 1 below, the present inventors have identified humanMBP amino acid residues Nos. 84-102 as one immunodominant domain ofhuman MBP recognized by a majority of peripheral T-cells isolated frompatients suffering from MS. Additional experimentation has determinedthat the immunodominant epitope within this domain is further localizedwithin human MBP amino acids Nos. 85-99. These data are presented inExample 3. (It appears by inference from the data in Example 3, that theminimal human MBP fragment within which the foregoing human hostimmunodominant epitope may reside is fragment 87-98 for some T-cellclones. But all T-cell clones reactive with 84-102 recognize thefragment 85-99.) A similar experiment with MBP fragment 143-168 can leadreadily to identification of the precise locus of that immunodominantepitope.

Experiments involving the animal model of MS, EAE, have shown thatprotein fragments including corresponding immunodominant epitopes ofguinea pig MBP and bovine MBP in rats, when administered orally toanimals suffering from the disease, are effective in the suppression ofthe symptoms of the disease (related patent application Ser. No.07/596,936 and Example 2 below) although some noninducing fragments aremore potent suppressors than inducing fragments. Further, it has beenshown that this orally effected suppression is due to the induction ofCD8⁺ suppressor T-cells (Lider et al., J. of Immunol. 142:748, 1989).Some experiments have been conducted by others in animals usingencephalitogenic fragments of MBP: See, e.g., Swierkosz, J. E., 1977, J.Immunol. 119:1501-1506; Su, X-M et al., 1991, J. Neuroimmunol.34:181-190 (i.v. use of MBP fragments--determined to be encephalitogenicin mice--coupled to spleen cells to abate adoptively transferred EAE inmice); Avrilionis, K. et al., 1991, J. Neuroimmunol. 35:201-210 (i.v.use in guinea pigs of liposo human MBP peptide fragment--shown to beencephalitogenic when administered to guinea pig--to suppress EAEinduced with the same fragment and i.p. and s.c. use of the free peptidefor the same purpose).

Thus, the peptides of the present invention can be advantageously usedin the design of specific immunosuppressive preparations containing suchpeptides which are in turn useful for the suppression of deleteriousT-cell proliferation. For example, peptides comprising sequences of thehuman MBP shown to induce anergy in human MBP-specific CD4⁺ T-cells orto induce T-suppressor cells specific for demyelination can beconstructed and used for such purposes. See, e.g., Examples 1 and 2below. Further, the results reported in Example 2 indicate that themethod and protocol of administration of the tolerizing agents affectthe mechanism which brings about the suppression of the autoimmunereaction. Thus, peptide fragments incorporating an immunodominantepitope of human MBP in humans are effective in inhibiting proliferationof MBP-reactive CD4⁺ human T-cells in vitro and are anticipated to beeffective by the same mechanism when administered via i.v. route inhumans. The same epitopic peptides are anticipated to be effective ininducing suppression of autoimmune attack of human neural tissue whenadministered to humans orally. The present inventors also have evidencethat a whole MBP (which encompasses the foregoing two immunodominantepitope regions) can induce suppression via elicitation of suppressorT-cells (active suppression) when MBP is orally administered in smallamounts and in multiple doses. The same antigen, also administeredorally but in high amounts and a single dose will induce suppression viaanergy. Finally, there is evidence that both mechanisms of suppressioncan be triggered by adjustment of the oral administration protocol fromMBP between the two extremes identified above.

Without wishing to be bound by theory, it is believed that the oral orenteral administration of immunodominant fragments of MBP can causesuppressor T-cells to be elicited that in turn suppress the T-cells thatcontribute to the autoimmune attack of a neural tissue (i.e., the brain,spinal cord, peripheral nerves or associated cell types). Nerve tissuedamage constituting the pathology seen in patients suffering from MS isbelieved to be the direct result of such an autoimmune attack. As thistolerizing mechanism involves the active induction of regulatory(suppressor) T-cells responsible for the suppression of theimmunoreactivity of cells in the vicinity of tissue under immune attack,it is an example of active suppression.

The present inventors have accumulated a large body of experimentalevidence that active suppression takes place by the elicitation oftolerizing-antigen specific T-suppressor cells which are targeted to thelocus (locuses) of the body where the antigen to which theseT-suppressors are specific can be found. This locus includes the tissueunder autoimmune attack. Once they encounter this antigen, theT-suppressors secrete suppressive cytokines such as the non-specificimmunosuppressive factor TGF-β, and interleukin-4 (IL-4) which suppressautoimmune responses including autoimmune attack. (See related patentapplication Ser. No. 843,752.)

In contrast, intravenous administration (or subcutaneous, orintraperitoneal administration) of the MBP fragments incorporatingimmunodominant epitopes is believed to bring about immune suppressionthrough another mechanism known as clonal anergy. Clonal anergy, orT-cell unresponsiveness, has been attributed to antigen presentation inthe absence of the appropriate co-stimulatory factors. Jenkins. M. K.PNAS. 84:5409-5413, 1987. The exact identity of the factors involved isill-defined, but soluble cytokines (e.g. B-7, EDCI, and an appropriateintracellular calcium concentration) have been implicated. More recentevidence, however, suggests that so-called "negative signals" ratherthan the absence of co-stimulatory factors are responsible for anergy.However, these signals have not yet been defined. See, LaSalle J. M. etal, J. Exp. Med., 176:177-186, June 1992. Rather than inducing theT-cell clones to proliferate, presentation of antigen by the APCswithout the co-stimulatory factors (and/or in the presence of thenegative signals) causes the T-cells to become unresponsive tosubsequent antigen stimulation, while remaining responsive to IL-2, andare thus described as being anergized (Jenkins et al., Proc. Natl. Acad.Sci. 84:5409, 1987; Mueller et al. Ann. Rev. Immunol. 7:455, 1989;Schwartz et al., Science 248:1349, 1990). Thus, autoimmuneresponse-promoting clones specific to an autoantigen such as MBP, willno longer proliferate in response to that antigen, reducing the immuneattack reactions which cause the tissue damage responsible for theautoimmune disease symptoms, such the neural tissue damage observed inMS.

Suppression by clonal anergy can be differentiated (and has been sodifferentiated in the experiments below) from active suppression byadoptive transfer experiments which test the ability or inability ofsuppressor T-cells transferred from a tolerized animal to a nontolerizedanimal to bring about suppression of immune function in the latteranimal. T-cell transfer brings about suppression if the suppressionmechanism is active, i.e. if it involves elicitation of T-suppressorcells and does not occur if the suppression mechanism is passive, i.e.if clonal anergy is involved. The results reported in Example 2illustrate instances in which each mechanism of immune suppression isinvolved and show that the suppression mechanism depends on one or moreof the following: (i) on the substance being administered to inducetolerance (for example, immunodominant epitopic fragments of MBP induceactive suppression via the oral route and energy via the parenteralroute); (ii) on the route of administration of the tolerizing antigen(for example, only epitopes that are recognized by attack T-cells induceanergy via i.v. route); and (iii) on the amount and frequency ofadministration (for example, orally administered MBP induces activesuppression when given in small amounts and multiple doses and passivesuppression when given orally in large amounts and a single dose).

The data show that both encephalitogenic and nonencephalitogenicfragments of MBP can elicit active suppression when orally administered,with those non-encephalitogenic fragments which incorporate animmunosuppressive epitope working only via active suppression and onlywhen administered via the oral route. The encephalitogenic fragments(incorporating an immunodominant epitope) may also elicit anergy via theoral route if administered in high amounts and single doses.

These results have important ramifications on the design of tolerizingagents and methods based on MBP as a tolerizer. Thus, depending on thetype of immune suppression desired, particular methods ofadministration, as well as particular fragments, may be used. Forexample, it may be desirable to use one or more disease-propagatingepitopic peptides via the i.v. or s.c. or i.p. route and (concurrently)one or more immunosuppressive epitopic peptides via the oral route.

It is also anticipated that tolerizing methods involving combinations ofadministration routes, and/or protocols and/or autoantigen fragments mayprove to be most effective. As will be understood by those skilled inthe art, the effectiveness of a fragment or combination of fragments (ora combination of a fragment and whole antigen) and the effectiveness ofa method (or combination of-methods) of administration of a MBP fragmentis a function of the age, sex, weight, physical condition, and diseasestage of the subject to be treated, and the concurrent use or absence ofother fragments or treatments. Consequently, the fragment(s) used andthe method of administration must be determined in substantial partbased on these factors, and may need to be determined experimentally ona case by case basis. Such determination, however, requires no more thanroutine experimentation, given the examples and guidelines containedherein.

Peptides based on the sequences of human MBP for use in the presentinvention, as identified in Example 1, can be synthesized usingwell-known solid phase methods (Merrifield, R. B. Fed. Proc. Soc. Ex.Biol., 21: 412, 1962 and J. Am. Chem. Soc. 85:2149, 1963; R. Mitchell A.R. et al, J. Am. Chem. Soc. 98:7357, 1976; Tam, J. et al., J. Am. Chem.Soc. 105:6442, 1983), preferably using a commercially available peptidesynthesis apparatus (such as that made by Applied Biosystems, FosterCity, Calif.) and following the manufacturers' instructions.Alternatively, such peptides can be synthesized by recombinant DNAtechniques as is now well-known in the art (Maniatis et al., MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratories, N.Y.,1982, see pp. 51-54 and pp. 412-30). For example, these peptides can beobtained as DNA expression products after incorporation of DNA sequencesencoding the desired fragment of MBP isolated from the desired speciesinto expression vectors and introduction of such vectors into suitableeukaryotic or prokaryotic hosts that will express the desired peptidesindividually or as part of fusion peptides or proteins, from which theycan be later isolated using well-known techniques.

Peptide analogs can be designed using the known amino acid sequencesencoded by the human MBP gene as disclosed below, using the synthetic orrecombinant techniques described above and the methods of, e.g., Eyler,E. B., in Advances in Experimental Medicine and Biology 21: 259-281,1978. For example, a peptide having a sequence based upon but differingfrom the exact amino acid sequence of the desired fragment of MBP can bechemically synthesized using the above-described techniques. Such apeptide can be tested for its effect on MBP-reactive CD4⁺ T-cells usinge.g. the procedure described in Example 1 for identifying a more preciselocation within-amino acids 84-102 for this particular immunodominantepitope-of human MBP or the procedure described in Example 4 for testingbinding to the T-cell receptor and to the MHC. An MBP-based peptide canbe tested in vitro for effectiveness orally in humans by exposingcollected, isolated peripheral MBP-specific suppressor T-cells fromindividuals to the peptide to determine whether they proliferate.Additionally, or alternatively, these isolated suppressor T-cells can betested to determine whether they release suppressive cytokines such asTGF-β and/or IL-4 upon exposure to an MBP peptide. (See, e.g. the use ofthe transwell system in co-pending U.S. application Ser. No. 843,752corresponding to PCT US92/01705 except that the use of spleen cells asAPC's is not necessary; the MBP peptide can be used to induce the cellsto release TGF-β.) MBP specific T-suppressor cells can be isolated byexposure to MBP and assessment of proliferation followed by bindingstudies using anti-CD8⁺ antibody.

The present invention also provides pharmaceutical formulations anddosage forms for oral or parenteral use in the suppression of autoimmuneattack T-cell function in humans, particularly those subjects sufferingfrom MS. In general such dosage forms contain one or more peptidesaccording to the invention which are fragments of human MBP and analogsthereof, in an amount effective to suppress proliferation of immuneattack cells. Suppression of function which results in an in vitrosuppression of immune attack cells, such as MBP-specific CD4⁺ T-cellsand/or attenuation of one or more symptoms of MS in a patient that hasbeen treated pursuant to the method of the present invention isconsidered to be within the scope of the invention. See definitionssection above for what constitutes suppression and symptoms attenuation.

The T-cell suppressive peptides of the present invention may alsoencompass additional non MBP-derived amino acid sequences leading orfollowing the MBP-based sequences as long as these additional sequencesdo not defeat the suppressive function of such peptides. Testing of suchconstructs for immunosuppressive activity can be easily done using, forexample, one or more of the assay methods described herein.

The pharmaceutical formulations of the present invention may include, asoptional ingredients, pharmaceutically acceptable vehicles, carriers,diluents, solubilizing or emulsifying agents, and salts of the type thatare well-known in the art. Nonlimiting examples of such substancesinclude 0.5N saline in distilled water for parenteral use and lactosefor oral use.

The fragments of human MBP can be administered orally or by-inhalationin conjunction with synergists which may enhance the effectiveness ofthe immune suppression. Non-limiting examples of synergists for use inthe present invention include bacterial lipopolysaccharides from a widevariety of gram negative bacteria such as various subtypes of E. coliand Salmonella (LPS, Sigma Chemical Co., St. Louis, Mo.; Difco, Detroit,Mich.; BIOMOL Res. Labs., Plymouth, Pa.), Lipid A (Sigma Chemical Co.,St. Louis, Mo.; ICN Biochemicals, Cleveland, Ohio; Polysciences, Inc.,Warrington, Pa.) and immunoregulatory lipoproteins, such as peptidescovalently linked to tripalmitoyl-S-glycarylcysteinyl-seryl-serine (P₃C55) which can be obtained as disclosed in Deres, K. et al. (Nature,342:561-564, 1989) or "Braun's" lipoprotein from E. coli which can beobtained as disclosed in Braun, V., Biochim. Biophys. Acta 435:335-337,1976. LPS is preferred and Lipid A particularly preferred. Lipid A isparticularly preferred for use in the present invention because it isless toxic than the entire LPS molecule. LPS for use in the presentinvention can be extracted from gram-negative bacteria and purifiedusing the method of Galanes et al. (Eur. J. Biochem. 9:245, 1969) andSkelly, R. R., et al. (Infect. Immun. 23:287, 1979).

The effective amount of a synergist, e.g. LPS or Lipid A, to beadministered in conjunction with the MBP fragment broadly ranges betweenabout 0.15 and 15 mg per kg body weight of said mammal per day andpreferably between about 0.3 and 12 mg per kg body weight of said mammalper day.

The route of administration of the suppressive agents of the presentinvention is in an oral or parenteral form or combinations thereof. Oraladministration includes oral, enteral or intragastric administrationwith oral being preferred. Parenteral administration includesintraperitoneal, subcutaneous, intradermal and most preferablyintravenous administration routes.

In general, the MBP-based peptide or analog is administered orally to ahuman patient in an amount ranging between about 10 μg and about 20 mgper administration. preferably between about 100 μg and 250 μg. Theamount is pulse-administered in a single dosage form or multiple dosageforms. For whole MBP to elicit active suppression a dosage of 1 mg fivetimes daily is an example of an effective amount. For anergy, 10-20 mgof MBP parenterally are examples of an effective amount. Monitoring ofthe patient is desirable in order to optimize the dosage and frequencyof administration. The exact amount and frequency of administration to apatient may vary depending on the stage, frequency of manifestation andseverity of the patient's disease and the physical condition of thepatient, as is well-appreciated in the art. Such optimization ispreferably effected on a case-by-case basis. Optimization of the dosagenecessary for immune suppression does not represent undueexperimentation, given the guidelines disclosed herein.

In an alternative preferred embodiment of the present inventionpharmaceutical oral formulations or dosage forms according to thepresent invention can also be administered by inhalation, preferably inaerosol form. The inhalation mode of administration is preferably notthrough the nasal passages but through the bronchial and pulmonarymucosa. The MBP fragment and related compounds of the present inventioncan be administered to humans as dry powder particles or as an atomizedaqueous solution suspended in a carrier gas (e.g. air or N₂). Preferredaerosol pharmaceutical formulations may comprise for example, aphysiologically acceptable buffered saline solution.

The methods of the present invention may involve by-inhalationadministration of pharmaceutical formulations in the form of an aerosolspray using for example, a nebulizer such as those described in U.S.Pat. No. 4,624,251 issued Nov. 25, 1986; U.S. Pat. No. 3,703,173 issuedNov. 21, 1972; U.S. Pat. No. 3,561,444 issued Feb. 9, 1971 and U.S. Pat.No. 4,635,627 issued Jan. 13, 1971. The aerosol material is inhaled bythe subject to be treated.

Other systems of aerosol delivery, such as the pressurized metered doseinhaler (MDI) and the dry powder inhaler as disclosed in Newman, S. P.in Aerosols and the Lung, Clarke, S. W. and Davia, D. eds. pp. 197-224,Butterworths, London, England, 1984, can be used when practicing thepresent invention.

Aerosol delivery systems of the type disclosed herein are available fromnumerous commercial sources including Fisons Corporation (Bedford,Mass.), Schering Corp. (Kenilworth, N.J.) and American Pharmoseal Co.(Valencia, Calif.).

It is expected that lower amounts of the fragment of MBP of the presentinvention will be required using aerosol administration for treatment asthis effect has been found when treating EAE with whole MBP and adjuvantarthritis with collagen as disclosed in co-pending U.S. patentapplication Ser. No. 454,486 filed Dec. 20, 1989. Further, it appearsthat the immune suppression induced by inhalation of the fragment of MBPoccurs through the active suppression mechanism, similar to oraladministration. Weiner, H. L. et al FASEB 4(7):2102, 1990. The amountsof the fragment of MBP of the present invention which may beadministered in an aerosol dosage form would be between about 0.015 mgand about 1.5 mg per kg body weight of a mammal per day and mayoptionally include a synergist in amounts ranging between about 0.05 andabout 15 mg per kg body weight of said mammal per day and may beadministered in single dosage form or multiple dosage forms. The exactamount to be administered will vary depending on the state and severityof a patient's disease and the physical condition of the patient.

Dosage forms for parenteral administration will generally contain fromabout 1 to about 200 mg of a peptide according to the present inventionper dose per person and preferably about 10 mg to about 100 mg. Theforegoing description of inert optional ingredients and fine-tuning ofamounts and administration scheduling given above with respect to oralformulations pertain to parenteral formulations only.

It will be appreciated that the unit content of active ingredient oringredients contained in an individual oral or parenteral dose of eachdosage form need not in itself constitute an effective amount fortreating MS since the necessary effective amount can be reached byadministration of a plurality of dosage units.

The techniques described below in Examples 1-3 can be used to monitorthe effectiveness of the methods of the present invention and optimizethe amount and frequency of administration of the disease suppressiveagents of the present invention, as well as the fragments and method ofadministration used.

T-cells can be isolated from a patient's peripheral blood orcerebrospinal fluid, amplified and cloned as described in Examples 1-3(before and/or after treatment according to the present invention).Antibodies (either polyclonal or monoclonal) can be obtained directedagainst the MBP peptides of the present invention (using conventionaltechniques well known and used in the art) to assay for the presence ofMBP-reactive T-cells in a patient's peripheral blood and morespecifically for the presence of activated MBP-reactive CD4⁺ T-cellsbefore and/or after treatment according to the present invention. Thepeptides of the present invention are also useful in identifyingindividuals with T-cells reactive with MBP, using the method of Example1.

The present invention is described further below in specific workingexamples which are intended to illustrate the present invention withoutlimiting its scope.

EXAMPLE 1 Identification of the Major Immunodominant Region of Human MBP

MBP was extracted from human brain tissue and purified on a CM-52 column(Supplier: Wattman Biosystems Ltd Maidstone, Kent, U. K.) using thehighest molecular weight peak (18 kD) as described (Chou, F. C.-H. etal., J. Biol. Chem. 251:2671, 1976). MBP peptides were synthesized usinga solid phase method or were obtained from a commercial laboratory(Biosearch Lab Inc., San Raphael, Calif. ) and were purified by highpressure liquid chromatography as follows: Each peptide containingpreparation was made up in 0.1% trifluoroacetic acid (TFA) at 1 mg/ml.It was then processed in an HPLC column (Rainin Reverse Phase C4 or C18)using a 0-70% acetonitrile gradient containing 0.1% TFA. Peaks weredetected at 214 nm. The MBP peptide fragments used are set forth belowin Table 1. However, the sequence of human MBP is published. Therefore,only the numbers designating the amino acid residues contained in eachpeptide are necessary.

                                      TABLE 1                                     __________________________________________________________________________    Human MBP            Human MBP                                                Amino Acid           Amino Acid                                               Residues                                                                            Sequence       Residues                                                                            Seauence                                           __________________________________________________________________________    1-20: ASQKRPSQRHGSKYLATAST                                                                         11-30:                                                                              GSKYLATASTMDHARHGFLP                               21-40:                                                                              MDHARBGFLPRHRDTGILDS                                                                         31-50:                                                                              RHRDTGILDSIGRFFGGDRG                               41-60:                                                                              IGRFFGGDRGAPKRGSGKDS                                                                         51-70:                                                                              APKRGSGKDSHEPARTABYG                               61-82:                                                                              HHPARTAHYGSLPQKSEGRT                                                                         71-92:                                                                              SLPQKSEGRTQDENPVVHFF                               84-102:                                                                             DENPVVHFFKNIVTPRTPP                                                                          93-112:                                                                             KNIVTPRTPPPSQGKGRGLS                               113-132:                                                                            LSRFSWGAEGQRPGFGYGGR                                                                         124-142:                                                                            RPGFGYGGRASDYKSAHKG                                143-168:                                                                            FKGVDAQGTLSKIFKLGGRD                                                    __________________________________________________________________________

A rapid T-cell cloning technique was used to examine whether there wereimmunodominant epitopes on human MBP reactive with Class II MHCphenotypes and the frequency of such reactivity. A total of 15,824 shortterm T-cell lines were generated from 51 human subjects by culturingperipheral blood mononuclear cells (PMN) with purified MBP (100 μg)followed 3 days later, and then every 3-4 days, by the addition ofinterleukin-2 (IL-2; from ABI, Columbia, Md.) and 2 units recombinantinterleukin-4 (IL-4; Genzyme, Boston, Mass.). On Day 13 of culture, analiquot from each line was tested for reactivity to MBP using thefollowing proliferation assay: T-cell clones (1×10⁵ cells/well) wereplated in triplicate and cocultured with appropriate stimuli (i.e. 100μg whole MBP or 5% IL-2) for 72 hours at 37° C., 90% humidity, 5% CO₂,in 96 well flat bottomed microtiter plates (Costar). The cells werepulsed with 2 μCi [³ H]TdR (2 Ci/mmole, New England Nuclear, Boston,Mass.) for the last 18 hours of culture. APCs were prepared by pulsinghuman Epstein-Barr virus transformed human B-cells (the virus beingcommercially available from ATCC) or L-cells which are mouse cellstransfected with human DR₂ (L-cells are commercially available from ATCCunder accession No. ATCC-CCL1 and can be transfected according to themethod of Wilkinson, D. et al, J. Exp. Med., 1988, 167:1442-1458using-DNA encoding DR2 as per Wu, S. et al J. Immunol., 1987, 138:2953).B-cells or L-cells were used at 1×10⁶ cells/well in complete mediaeither in the presence or absence of 40 pM MBP or phospholipid protein(PLP) for 2 hours at 37° C., washing twice with 4° C. Hanks (Whittaker),followed by irradiation with 5000 rad. at 4° C. To stimulate T-cellswithout accessory APCS, 2 μM of MBP, PLP, or the appropriate fragmentwas added directly to the cells for the duration of the culture: 48hours, followed by pulsing thymogen and then harvesting. Ten thousandAPC's were used for 100,000 T-cell clones.

T-cell lines shown to be reactive to MBP or PLP were then tested usingthe same technique for reactivity to overlapping oligopeptide 20-mersencompassing the human MBP sequence as shown in Table 1 above. MBP andPLP reactivity frequency analysis was performed on patients withdefinite, relapsing-remitting MS (as diagnosed by Magnetic ResonanceImaging (MRI) and clinical examination), as well as on subjects withother neurologic diseases and on normal subjects (all age- andsex-matched to the MS patients). The results are shown in Table 2 below.

                                      TABLE 2                                     __________________________________________________________________________                SEX #Ag REACTIVE LINES                                                                         MEAN FREQUENCY OF                                            (%) TOTAL # LINES                                                                              Ag REACTIVE LINES                                AGE         (M/F)                                                                             MBP   PLP    MBP    PLP                                       __________________________________________________________________________    MS*   34.2 ± 1.4                                                                       35/65                                                                             554/7746                                                                            20/432 7.18 ± 2.38                                                                       3.34 ± 1.56                            (n = 23)                                                                      OTHER 38.7 ± 3.2                                                                       43/57                                                                             118/2880                                                                            3/384  4.10 ± 1.04                                                                       0.90 ± 0.62                            N* DIS-                                                                       EASE                                                                          (n = 10)                                                                      NORMAL                                                                              30.3 ± 1.5                                                                       50/50                                                                             73/1742                                                                             ND     4.70 ± 1.58                                                                       ND                                        (n = 6)                                                                       __________________________________________________________________________     *MS = Multiple Sclerosis                                                      **N = Neurological                                                       

Patients with MS were Caucasian and had well-characterized relapsingremitting disease with at least two exacerbations within the previous 24months and positive lesions as seen using MRI at the time of blooddrawing. Subjects with other central nervous system diseases had thefollowing diagnoses: 1-3 weeks after either cerebrovascular accident(n=4), brain trauma with CNS hemorrhage (n=4), or metastatic brain tumor(n=2). The total number of T-cell lines reactive with MBP or PLP and thetotal number of T-cell lines generated are shown in Table 2 ("Ag" means"antigen"). In addition, the frequency of MBP and PLP-reactive lines wascalculated separately for each subject by dividing the number ofMBP-reactive lines by the total number of lines generated and the meanvalues +/- SEM are given. The same conclusions can be drawn regardingreactivity to PLP although to a lesser extent than reactivity to MBP orits fragments.

While the frequency of lines reactive to all fragments of MBP wasslightly higher in subjects with MS as compared to the other subjects,this increase was not statistically significant. However, as discussedbelow, a significantly greater number of the MBP reactive cells linesfrom MS patients were reactive with the fragments including amino acids84-102 and 143-168, respectively, thus identifying these peptides andthe corresponding fragments of MBP as containing immunodominant epitopesof MBP active in the development of MS.

Of a total of 302 cell lines from patients with MS that could beexpanded and confirmed to react with MBP on repeated analysis, 140(46.4%) reacted with MBP residues 84-102; and approximately 70-80%reacted with either MBP residues 84-102 or 143-168. In the controlgroups, 11 of a total of 100 MBP reactive T-cell lines (11.0%)recognized the 84-102 peptide and about 34% recognized either the 84-102or the 143-168 peptide. The actual frequency of T-cells derived from theperipheral blood that reacted with each MBP peptide for each individualsubject was calculated. The mean values for patients with MS and thecontrol subjects are shown in the rightmost column of Table 2. Thecorresponding immunodominant peptide(s) of PLP can be identified by thesame methods as described herein for the MBP peptides.

The frequency of MBP-peptide specific cell lines from normal subjectsand other neurologic disease controls were virtually identical and thuscombined for analysis. The mean frequency of T-cell lines from subjectswith MS that were selectively reactive to MBP residues 84-102 was higheras compared with controls (FIG. 1). Significant but less strikingincreases in reactivity to MBP residues 61-82 and 124-142 were alsoobserved in MS patients, while both MS and control subjects showed highfrequencies of T-cell lines reactive with MBP residues 143-168. IL-2 wasused to select the T-cell lines that were activated. After primarystimulation with IL-2 the thus activated cell lines recognized MBPpeptides.

Expansion of these findings into a larger population of MS patients wasdone, using the techniques described above. An additional 132 T-celllines (63 from MS patients and 88 from normal controls) were studied,with the results reported in Table 3, below. In summary, these resultssupport the conclusion of peptides containing MBP amino acid residues84-102 and 143-168 as the immunodominant domains of MBP.

                  TABLE 3                                                         ______________________________________                                                        No. of                                                                        MBP-Peptide  Peptide Reactivity                                               Reactive     (Fraction of                                     Subject                                                                              Stimulus Lines        MBP-Reactive Lines)                              ______________________________________                                        MS-HY  MBP      17           84-102 (17/17)                                          IL-2     4            84-102 (4/4)                                     MS-SW  MBP      8            84-102 (2/8)                                                                  143-158                                                                              (5/8)                                                                  other  (1/8)                                            IL-2     3            143-168                                                                              (2/3)                                                                  other  (1/3)                                     MS-CY  MBP      14           143-168                                                                              (9/14)                                                                 other  (5/14)                                           IL-2     4            143-168                                                                              (3/4)                                                                  other  (1/4)                                     MS-HK  MBP      7            143-168                                                                              (6/7)                                                                  other  (1/7)                                            IL-2     6            143-168                                                                              (5/6)                                                                  other  (1/6)                                     MS-JA  MBP      10           84-102 (1/10)                                                                 143-168                                                                              (8/10)                                           IL-2     4            143-168                                                                              (4/4)                                     MS-MI  MBP      5            84-102 (3/5)                                                                  143-168                                                                              (1/5)                                                                  other  (1/5)                                            IL-2     2            84-102 (1/2)                                                                  143-168                                                                              (1/2)                                     MS-AN  MBP      2            143-168                                                                              (2/2)                                            IL-2     2            143-168                                                                              (2/2)                                     MS-ST  MBP      nd           nd                                                      IL-2     18           84-102 (6/18)                                                                 143-168                                                                              (6/18)                                                                 93-142 (3/18)                                                                 other  (3/18)                                    NS-kw  MBP      8            84-102 (2/8)                                                                  143-168                                                                              (5/8)                                                                  other  (1/8)                                            IL-2     1                                                                                          84-102 (100%)                                    NS-jl  MBP      12           84-102 (10/12)                                                                other  (2/12)                                           IL-2     12           84-102 (7/12)                                                                 143-168                                                                              (5/12)                                    NS-aa  MBP      2            84-102 (2/2)                                            IL-2     2            84-102 (2/2)                                     NS-dd  MBP      12           84-192 (4/12)                                                                 143-168                                                                              (6/12)                                                                 other  (2/12)                                           IL-2     2            84-102 (1/2)                                                                  143-168                                                                              (1/2)                                     NS-nb  MBP      37           84-102 (37/37)                                          IL-2     2            84-102 (2/2)                                     ______________________________________                                         nd = not done                                                            

                                      TABLE 4                                     __________________________________________________________________________               frequency of IL-2 responsive                                                                  frequency of IL-2 responsive                       CSF        T-cells × 10.sup.-4                                                                     T-cell × 10.sup.-4                           subject                                                                             cells/nm.sup.3                                                                     CSF     PBMC    CSF     PBMC                                       __________________________________________________________________________    patients   (A)     (B)     (C)     (D)                                        with MS                                                                       MS-1  12.2 18.0 (13.1-25.7).sup.a                                                                4.7 (2.3-8.2)                                                                         2.6 (1.8-6.6)                                                                         N.D..sup.b                                 MS-2  1.5  10.4 (6.1-14.7)                                                                       6.7 (4.2-11.8)                                                                        5.5 (4.3-9.2)                                                                         N.D.                                       MS-3  1.2  7.7 (4.3-12.8)                                                                        4.7 (2.9-8.8)                                                                         3.8 (2.2-7.2)                                                                         N.D.                                       MS-4  1.2  16.6 (11.5-23.7)                                                                      13.3 (9.9-19.7)                                                                       9.1 (6.9-14.8)                                                                        N.D.                                       MS-5  1.5  17.5 (13.1-24.2)                                                                      11.2 (7.8-17.2)                                                                       8.3 (5.3-14.5)                                                                        N.D.                                       MS-6  2.0  23.3 (16.9-32.4)                                                                      8.7 (6.4-13.2)                                                                        20.0 (14.8-28.0)                                                                      N.D.                                       MS-7  3.4  25.0 (18.7-33.1)                                                                      7.7 (4.2-12.4)                                                                        8.4 (6.0-13.5)                                                                        1.8 (1.4-3.2)                              MS-8  2.8  7.7 (4.3-11.9)                                                                        2.3 (1.7-5.8)                                                                         6.7 (4.2-11.8)                                                                        1.9 (1.3-4.4)                              MS-9  5.0  12.1 (8.6-17.8)                                                                       12.1 (8.8-17.4)                                                                       1.7 (1.2-4.3)                                                                         N.D.                                       MS-10 4.8  24.3 (17.2-32.2)                                                                      2.4 (6.4-13.5)                                                                        3.4 (2.3-7.4)                                      MS-11 5.7  23.8 (16.8-32.5)                                                                      3.2 (2.2-7.8)                                                                         4.9 (2.9-8.6)                                                                         N.D.                                       MS-12 4.6  6.1 (3.6-11.1)                                                                        2.7 (1.8-5.6)                                                                         N.D.    N.D.                                       MS-13 2.2  17.8 (13.2-24.3)                                                                      10.0 (7.8-15.4)                                                                       N.D.    N.D.                                       MS-14 3.4  25.0 (18.7-33.5)                                                                      16.6 (11.5-23.5)                                                                      2.4 (1.7-6.9)                                                                         N.D.                                       MS-15 3.8  18.2 (13.4-24.8)                                                                      8.3 (5.8-11.4)                                                                        6.6 (5.2-10.8)                                                                        N.D.                                       MS-16 4.5  6.2 (4.3-11.8)                                                                        4.2 (2.4-7.5)                                                                         6.4 (5.2-10.7)                                                                        N.D.                                       MS-17 2.6  12.5 (8.6-18.3)                                                                       6.2 (4.6-10.8)                                                                        1.4 (1.3-4.5)                                      MS-18 1.2  7.7 (4.2-13.1)                                                                        4.3 (2.7-9.2)                                                                         N.D.    N.D.                                       MS-19 4.6  11.1 (7.6-17.0)                                                                       2.0 (1.4-5.6)                                                                         16.5 (11.5-24.1)                                                                      2.7 (1.8-5.2)                              MS-20 3.8  23.8 (16.9-32.0)                                                                      8.3 (6.0-12.9)                                                                        N.D.    N.D.                                       mean  3.60 15.7    7.8     5.4     0.32                                       patients   (E)     (F)     (G)     (H)                                        with CND                                                                      CND-1 5.0  10.4 (7.3-16.4)                                                                       9.6 (7.3-14.6)                                                                        N.D.    N.D.                                       CND-2 2.0  7.2 (5.1-10.8)                                                                        8.3 (5.8-12.4)                                                                        N.D.    N.D.                                       CND-3 2.4  4.6 (2.8-9.0)                                                                         7.8 (4.2-14.5)                                                                        N.D.    N.D.                                       CND-4 3.0  5.8 (4.6-10.1)                                                                        5.4 (4.2-9.9)                                                                         N.D.    N.D.                                       CND-5 5.0  4.1 (2.3-8.5)                                                                         8.7 (6.4-13.4)                                                                        N.D.    N.D.                                       CND-6 4.4  4.8 (2.9-9.2)                                                                         6.8 (5.2-10.2)                                                                        N.D.    N.D.                                       CND-7 1.2  3.3 (2.0-7.9)                                                                         4.2 (2.4-8.9)                                                                         N.D.    N.D.                                       CND-8 2.0  2.1 (1.3-5.0)                                                                         4.9 (3.8-9.1)                                                                         N.D.    N.D.                                       mean  3.13 5.2     6.9     --      --                                         column pair                                                                         (A)-(B)                                                                            (C)-(D) (E)-(F) (A)-(B) (B)-(F) (C)-(G)                            p value.sup.c                                                                       0.0001                                                                             0.0001  0.165   0.001   0.578  0.0001                              __________________________________________________________________________     .sup.a 95% confidence limits.                                                 .sup.b not detectable at the cell concentration used.                         .sup.c calculated by t-test.                                             

The foregoing results in Table 4 demonstrate that a dramatically highernumber of additional MBP-reactive T-cell clones can be identified in MSpatients (compared to controls) upon primary stimulation of MBP-specificT-cells with IL-2. This indicates that MBP-reactive T-cell clones can bede-activated (possibly deanergized) by exposure to IL-2, subsequent towhich they become reactive to MBP peptides.

EXAMPLE 2 Mechanism of Induction of Immune Tolerance

The experiments in this Example were done to compare the effectivenessof suppression of EAE using different fragments of MBP, to compare oraland intravenous administration of the protein fragment, and to comparetreatment of the disease state when it was induced oradoptively-transferred. Induced EAE occurs when MBP is used to immunizea host and is administered intramuscularly in conjunction with anadjuvant, while adoptively transferred disease occurs when anMBP-reactive cell line is activated then injected into the animal. (seeInduction of EAE section below for details.) In this example, thefollowing materials and methods were used.

Animals

Female Lewis rats 6-8 weeks of age were obtained from Harlan-SpragueDawley Inc. (Indianapolis, Ind.). Animals were housed in Harvard MedicalSchool Animal Care Facilities and maintained on standard laboratory chowand water ad libitum. Animals were maintained in accordance with theguidelines for the Committee on Care of Laboratory Animals of theLaboratory Research Council (Pub. #DHEW:NIH, 85-23, revised 1985).

Antigens and Reagents

Guinea pig MBP was purified from brain tissue by the modified method ofDeibler et al. (Prep. Biochem. 2:139, 1972). Protein content and puritywere checked by gel electrophoresis and amino acid analysis.Concanavalin A and histone were obtained from Sigma (St. Louis, Mo.).Peptides were synthesized in the peptide facility of the Center forNeurologic Disease, Brigham and Women's Hospital, and purified on HPLC.The amino acid sequences of the peptides synthesized are: 21-40,MDHARHGFLPRHRDTGILDS (immunosuppressive epitope region when orallyadministered to rats); 71-90, SLPQKSQRSQDENPVVHF (immunodominantencephalitogenic region in rats); 151-170, GTLSKIFKLGGRDSRS.

Induction of Tolerance

For oral tolerance or active suppression, rats were fed 1 mg of MBPdissolved in 1 ml PBS, or PBS alone, by gastric intubation with a18-gauge stainless steel animal feeding needle (Thomas Scientific,Swedesboro, N.J.). Animals were fed five times at intervals of 2-3 dayswith the last feeding two days before immunization. For intravenoustolerance or clonal anergy, rats were injected with 0.1 mg of MBP, MBPpeptides, or histone dissolved in 0.1 ml PBS, or PBS alone. Animals wereinjected via the ocular vein five times at intervals of 2-3 days withthe last injection two days before immunization.

Induction of EAE

For actively induced disease, Lewis rats were immunized in the left footpad with 25 μg of guinea pig MBP in 50 μl of PBS emulsified in an equalvolume of complete Freund's adjuvant (CFA) containing 4 mg/ml ofmycobacterium tuberculosis (Difco). For adoptively transferred EAE, anMBP active T cell line was established from rats immunized with MBP inCFA, raised and maintained according to the method of Ben-Nun et al.(Euro. J. Immunol. 11:195, 1982). Encephalitogenic cells were collectedafter activation by culture with Concanavalin A (ConA) (2 μm/ml) usingirradiated thymocytes from immunized animals as APCs. Cells wereharvested from cultures via a ficol hypaque gradient (Hypaque 1077,Sigma) and washed twice in PBS prior to transfer. 5×10⁶ encephalitogeniccells were injected intraperitoneally in 0.1 ml PBS into irradiated (750rads, 24 hrs. earlier), recipient rats. Cell viability of both modulatorand encephalitogenic cells was determined by trypan blue exclusion andwas greater than 90%. In all experiments 5 animals were used perexperimental group.

Purification of T Cell Subsets for Adoptive Transfer of ProtectionFollowing Oral Tolerization

Depletion of lymphocyte subsets was performed by negative selectionusing magnetic beads according to a modified method of Cruikshank et al.(J. Immunol. 138:3817, 1987). Spleen cells were incubated with a 1:10dilution of mouse anti-rat CD8 or CD4 monoclonal antibody (clones OX/8or W3/25 respectively, Serotec/Bioproducts, Indianapolis, Ind.), for 30minutes on ice, washed twice, and then added to prewashed magneticparticles, with an average diameter of 4.5 μm (M-450) with goatanti-mouse IgG covalently attached (Dynal, Fort Lee, N.J.). The quantityof magnetic beads used was calculated as being 10 times the estimatedtarget cell population. The cells were incubated with the beads in 0.5ml of RPMI 1640 medium supplemented with 10% fetal calf serum in a 10 mlround bottom test tube (Nunc) for 30 min. on ice with gentle shakingevery 5 min. After incubation, the bead/cell suspension was washed with5 ml of medium, and the cell-mAb-bead complexes were separated fromunlabeled cells in a strong magnetic field using a magnetic-particleconcentrator (Dynal-MPC-1) for 2 minutes. The supernatant was removed,and the procedure was repeated twice-to obtain the nonadherent fraction.The cells in the CD4⁺ and CD8⁺ depleted populations were >95%CD4⁺ CD8⁻or CD4⁻ CD8⁺, as demonstrated by indirect flow cytometry. Whole spleenpopulations from MBP fed or control animals were cultured (5×10⁶ cellsin 1 ml of proliferation media), in the presence of Con-A (2 μg/ml).Depleted populations were cultured at a concentration of 2.5×10⁶ cellsper ml. The resulting subsets were used as modulator cells.

Clinical Evaluation

Animals were evaluated in a blinded fashion every day for evidence ofEAE. Clinical severity of EAE was scored as follows: 0, no disease; 1limp tail; 2, hind limb paralysis; 3, hind limb paraplegia,incontinence; 4, tetraplegia; and 5 death. Duration of disease wasmeasured by counting the total number of days from disease onset(usually days 10 or 11 after active immunization and 3-5 days afteradoptive transfer of disease) until complete recovery for each animal.

Delayed Type Hypersensitivity (DTH) Testing

DTH was tested by injecting 25 μg of MBP in PBS subcutaneously in theear. Thickness was measured by a blinded observer, before and 48 hoursafter challenge, using micrometer calipers (Mitutoyo, Japan). Thedifference of ear thickness before and after challenge was recorded foreach animal, and the result was expressed as the mean for eachexperimental group±SEM.

Histology

Histologic analysis of pathological changes was performed in rats withadoptively transferred EAE. Spinal cords were removed on day 15 afteradoptive transfer and fixed with 10% neutral buffered formalin. Paraffinsections were prepared and stained with Luxol fast blue-hematoxylin andeosin, by standard procedures (Sobel et al., J. Immunol. 132:2393,1984). Spinal cord tissue was sampled in an identical manner for eachanimal and numbers of inflammatory foci per section (clusters of >20 ormore aggregated inflammatory cells), in parenchyma and meninges werescored in a blinded fashion (Sobel et al., supra).

Statistical Analysis

Clinical scales were analyzed with a two-tailed Wilcoxon rank sum testfor score samples, chi square analysis was used in comparing theincidence of disease between groups, and comparison of means wasperformed by using the Student's t-test. For individual experiments, 5animals were used per group.

Results

Suppression of Adoptively Transferred EAE by Oral Tolerization to MBP

To evaluate the effect of prior oral administration of MBP on adoptivelytransferred EAE, MBP-fed and control rats were intraperitoneallyinoculated with 5×10⁶ MBP-specific, Con-A stimulated, encephalitogenicline cells. MBP reactive cells were transferred 2 days after the lastfeeding. FIG. 2A graphs the clinical scores of animals which were orallytolerized to MBP and then inoculated with the MBP-specific cells (blackcircles) and compares them with the clinical scores of naive animalssimilarly inoculated (open circles). As shown in this figure, oraladministration of MBP had no effect on adoptively transferred EAE. Thepresent inventors propose that the failure of oral tolerization tosuppress adoptively transferred EAE is due to the fact that thetransplanted encephalitogenic T-cells are activated and able to migrateto the target organ rapidly wherein they initiate immune attack beforesufficient numbers of suppressor T-cells: migrate to the lymph nodes;and (iii) migrate to the target organ (brain). Thus, the ratio ofregulatory to encephalitogenic cells present at the target organ and thetiming of their entry appear to be critical.

However, adoptively transferred EAE was suppressed when spleen cellsfrom orally tolerized animals were co-transferred with theencephalitogenic cells to naive recipients (FIG. 2B), indicating thatalready-elicited suppressor T-cells can successfully prevent diseaseeven when they are co-administered with the immune attack cells.

FIG. 2B graphs the clinical scores of animals which were co-injectedwith 5×10⁶ encephalitogenic cells with 1.5×10⁶ spleen cells from animalsorally tolerized to MBP. For co-transfer, cells from orally tolerizedanimals were mixed with encephalitogenic cells and injected (blackcircles). As also shown in FIG. 2B, similar protection was observed whenencephalitogenic and modulator cells were injected separately in theright and left flanks (open circles), which indicates that theprotective effect is not due to interaction between suppressor T-cellsand attack T-cells. The clinical scores of positive control animals inFIG. 2B are indicated by black squares.

Suppression of Adoptively Transferred EAE is Dependent on Cb8⁺ T Cellsfrom Orally Tolerized Animals

To determine whether suppression of adoptively transferred EAE wasdependent on a specific T-cell subset, spleen cells from MBP fed animalswere depleted on CD4⁺ or CD8⁺ T-cell subsets prior to adoptive transferand used as modulators. As shown in FIG. 3, adoptive transfer ofprotection was abrogated by transfer of CD8⁺ depleted spleen cells, butnot by transfer of CD4⁺ depleted spleen cells (mean maximalscore=2.3±0.2 vs. 0.7±0.2, respectively, p<0.01). Open squares:unselected spleen cell population, black circles CD4⁺ depleted spleencells; open circles CD8⁺ depleted spleen cells.

Delayed Type Hypersensitivity (DTH) Responses Associated with AdoptivelyTransferred EAE

A correlation between DTH responses and the suppression of activelyinduced EAE following oral tolerance has been found (Miller et al., J.Exp. Med. 174:791, 1991; Miller et al., Proc. Natl. Acad. Sci. 89:421,1992). To determine whether a similar correlation existed in adoptivelytransferred EAE, DTH responses were measured. As shown in FIG. 4,prominent DTH responses developed in animals undergoing adoptivelytransferred EAE and DTH responses were suppressed by the co-transfer ofsplenocytes from animals orally tolerized to MBP. The suppressed DTHresponses were abrogated by depletion of CD8⁺, but not CD4⁺ T-cellsprior to transfer. (Δ ear swelling CD4⁺ depleted vs. CD8⁺depleted=0.6±0.1 vs. 1.8±0.2, p<0.01).

Effect of Co-transfer of Cells from MBP Orally Tolerized Animals on CNSHistology in Adoptively Transferred EAE

Oral administration of MBP suppresses CNS inflammation in activelyinduced EAE (Higgins et al., J. Immunol. 140:440, 1988). Nevertheless,not all immune specific immunomodulatory treatments of EAE that suppressclinical disease affect CNS inflammation (Offner et al., Science251:430, 1991). As shown in FIG. 5, there was decreased inflammation inboth the parenchyma and meninges when cells from MBP-fed animals weretransferred and this suppression was observed when CD4⁺ depleted, butnot CD8⁺ depleted modulator spleen cells from orally tolerized animalswere transferred. Number of CNS (parenchyma+meninges) inflammatory focifor the specific groups were as follows: Control=76±8.2; MBPfed=3.8±1.8; CD4⁺ depleted=2.8±1.0; CD8⁺ depleted=65±4; (p<0.01, MBP fedand CD4⁺ depleted vs. control or CD8⁺ depleted).

Suppression of Actively Induced and Adoptively Transferred EAE FollowingIV Administration of MBP

As shown in FIG. 6A, intravenous (IV) injection of MBP markedlysuppressed EAE actively induced by immunization with MBP/CFA (meanmaximal score=0.5±0.2, vs. control injected histone=3.0±0.3; p<0.01), inan analogous manner to suppression by oral tolerization with MBP. Incontrast to oral tolerization, however, which did not protect againstadoptively transferred EAE (FIG. 2A), IV injection of MBP alsosuppressed adoptively transferred EAE (mean maximal score=0.4±0.2, vs.control=3.2±0.2; p<0.01). (FIG. 6B) However, unlike oral tolerization,disease protection could not be adoptively transferred with spleen cellsfrom IV tolerized animals when such cells were co-transferred with anMBP encephalitogenic line (mean maximal score=2.8±0.2, vs. controlp=N.S. (FIG. 6B).

Suppression of EAE Following Oral or IV Administration of MBP Peptides

To further investigate the mechanism of oral vs. IV tolerance, MBPpeptides encompassing both encephalitogenic and non-encephalitogenicregions of MBP were administered both orally and intravenously prior toimmunization for actively induced disease. MBP peptide 71-90 of guineapig MBP is encephalitogenic in Lewis rats (Swanborg et al., J. Immunol.114:191, 1975). As shown in FIG. 7, suppression of EAE via IVtolerization only occurred with whole MBP and encephalitogenic peptide71-90, but not with guinea pig MBP peptide 21-40. Oral tolerization with21-40, however, was effective in suppressing EAE. Guinea-pig peptide21-40 was chosen as experiments demonstrated that it triggered TGF-βrelease from spleen cells of rats orally tolerized to whole MBP. Miller,A. et al. FASEB 6:1686, 1992. Control guinea pig MBP peptide 131-150 didnot suppress when administered either orally or intravenously. Of noteis that in addition to suppressing via the IV route, encephalitogenicMBP peptide 71-90 also suppressed when given orally. This resultindicates that peptides derived from the immunodominant domain of agiven MBP towards a given host can suppress T-cell function when theyare orally or intravenously administrated, but do so by differentmechanisms depending on the route and protocol of administration.

The results of these experiments show that there are basic differencesin the mechanism of suppression of EAE between orally and parenterally(e.g. intravenously) administered MBP. The results suggest that orallyadministered antigen acts predominantly via the generation of activesuppression, whereas parenterally administered antigen acts via clonalanergy. Specifically supporting this conclusion is the inability ofspleen cells from IV tolerized animals to suppressadoptively-transferred EAE. Additionally, different fragments of MBPwere more or less effective in suppression with the different routes ofadministration. (see, for example, FIG. 7) The present findings may beused to advantage in designing immunosuppressive methods based onantigen-driven tolerance, such as the method of the present invention.

EXAMPLE 3 Fine-Tuning of the Determination of the Immunodominant Epitopeof Human MBP in Human by Assessing Ability of Overlapping Peptides toStimulate MBP Specific T-Cell Clone

Using the cell proliferation assay described in Example 1 above, theability of a peptide consisting of the immunodominant domain of humanMBP (i.e. the sequence of amino acids 84-102) to stimulate T-cellproliferation was assessed and compared to that of a series of peptideshaving amino acid sequences representing N-terminal and C-terminalprogressive truncations of the immunodominant domain was determined. Asshown in FIG. 8, (and Table 5 below) the tested T-cell linesproliferated with a N-terminal truncation down to amino acid 85, afterwhich there was a dramatic decrease in ability of the fragment toactivate proliferation. Progressive C-terminal truncation quicklydrastically affects stimulation ability, where truncation to onlyresidue 99 does not substantially affect epitope function. In absence ofC-terminal truncation, as shown in FIG. 8, loss of amino acids 85 and 86also appear to be tolerated by the tested clone. In FIG. 9, fourdifferent T-cell clones were exposed to whole MBP 84-102 peptide and topeptides 85-99 and 86-97 to test whether different peptides causeddifferences in proliferation of the different clones. Althoughindividual clones have inherently different abilities to proliferate inthe presence of an epitopic peptide, nevertheless, the ability of aparticular peptide to cause a clone to proliferate is qualitativelysimilar from clone to clone. From these studies it appears that thefragment amino acids 85-99 would comprise a minimal immunodominantfragment able to stimulate T-cell activity for all T-cell clones.

Table 5, below lists the ability of human MBP (84-102) specific T-cellclones to proliferate in the presence of MBP (84-102) peptide andtruncated or modified versions of this peptide. The numbers in thematrix are peptide concentrations (expressed in micromolar) that give50% maximal stimulation of the T-cell clones. Maximal stimulation of theT-cell clones was assessed by exposing them to unmodified untruncatedMBP (84-102) peptide. The bold face designates "50" and ">50" mean afive-fold or more than a five-fold loss in stimulative activity comparedto the untruncated unmodified MBP (84-102) peptide.

                                      TABLE 5                                     __________________________________________________________________________    Comparison of the Relative Efficiency of Truncated Peptides                   to Stimulate MBP (84-102) Specific T cell clones                              Ob.1H8    Ob.1E10                                                                            Ob.2G9                                                                             Ob.1C3                                                                             Ob.1A12                                                                            Ob.2F3                                                                             Ob.3D1                                                                             Hy.2E11                               __________________________________________________________________________    84-102                                                                             3.1  4.0  3.8  2.9  1.6  2.1  2.0  0.26                                  84-102tyr                                                                          2.0  3.1  3.2  2.9  1.6  1.5  2.0  0.26                                  85-102                                                                             12   1.8  3.6  4.0  0.4  1.5  2.0  0.31                                  86-102                                                                             >50* >50  >50  50   4.8  17   2.0  0.45                                  87-102              >50  >50  >50  4.2  0.80                                  88-102                             >50  50                                    89-102                                  >50                                   84-100                                                                             2.2  1.8  1.2  2.2  0.65 1.9  2.5  0.08                                  84-99                                                                              3.1  4.0  2.1  2.3  1.6  2.1  2.2  0.26                                  84-97                                                                              >50  >50  >50  >50  22   22   11   25                                    84-96                    >50  >50  50   >50                                   84-95                              >50                                        __________________________________________________________________________

Peptide concentrations (μM) that give 50% maximal stimulation (referencepoint: MBP(84-102) peptide) are given. *More than a fivefold loss inactivity compared to the MBP(84-102) peptide.

EXAMPLE 4 T-Cell Recognition of the Alanine Analog Peptides of MBP85-99-Reactive T-Cell Clones

FIG. 11 shows the binding of each peptide to MHC (left column) and to aT-cell clone (right panel). DRB1*1501 transfected L cells present theimmunodominant MBP (84-102) peptide to autoreactive T-cells. APC's werepulsed with the MPB (84-102) peptide (100 μg/ml) for B-cell lines and 50μg/ml for DR transfectants, irradiated with 5000 rad and co-culturedwith the T-cell clones for three days followed by a thymidine pulse. Theresults (³ H-thymidine uptake compared to native peptide) are expressedin black rectangles (90% of the maximum stimulation) grey with crosses(>50% of the maximum stimulation) light grey (>10% of the maximumstimulation) and white (no activity). The results show that the Val atposition 898 and the Phe at position 92 are involved in MHC binding.Truncation data suggest that the isoleucine at position 95 is alsoinvolved in MHC binding. T-cell receptor contact points include His at90, Phe at 91 and Lys at 93.

T-cell clone Hy.2Ell will tolerate an Arg substitution in place of Lysat 93 but the Ob T-cell clones will not. The proposed binding motifs forDRB1*1501 and DRB5*0101 are in FIG. 10. The arrows up indicate bindingto the DR receptor and the arrows down indicate binding to the MHC ofAPC.

A series of analog peptides will be synthesized to both conservative andnon-conservative amino acid substitutions at positions 88-95 that willbe used. For example, the negative charge aspartic acid will besubstituted with another negatively charged residue (glutomic acid), anamino acid with the same bulk but no charge (asparginine), an amino acidwith the opposite charge (lys), and lastly a small amino acid such asala. Proximately 60 peptides will need to be synthesized.

Preliminary data suggest that the 148-162 region of MBP is the minimalepitope for dominant epitope 143-168. When the core region of thispeptide is defined using the methods outlined in Examples 2 and 3, asimilar analysis as outlined in this Example 4 for MBP peptides 85-99.

Analog peptides will be analyzed for DR26-(DRB1*1501), DR2a-(DRB5*0101),DR4, DR7, and DQ1.1 binding. This analysis will elucidate further whichamino acid residues are involved in MHC binding. Analog peptides thathave no less than 10-times the binding capacity of the native peptidewill be used to examine T-cell receptor specificity.

The present invention has been illustrated by reference to specificExamples. It will be apparent to those of ordinary skill, however, thatmany additions, deletions and modifications are possible withoutdeparting from the spirit or scope of the invention as claimed.

What is claimed:
 1. A pharmaceutical formulation comprising a peptidethe amino acid sequence of which is all or a segment of the sequenceSLPQKSHGRTQDENPVVHFFKNIVTPRTPPPSQGKGRGLS, provided that said segmentcomprises at least DENPVVHFFKNIVTPRTPP.
 2. A pharmaceutical formulationcomprising a peptide the amino acid sequence of which is all or asegment of the sequence SLPQKSHGRTQDENPVVHFFKNIVTPRTPPPSQGKGRGLS,provided that said segment comprises a portion of DENPVVHFFKNIVTPRTPPsufficient to impart to said peptide the property of stimulating, asdetermined by proliferation assay, the subgroup of T-cells of theDR2+type from remitting-relapsing multiple sclerosis patients that arereactive with another peptide the sequence of which consists ofDENPVVHFFKNIVTPRTPP, said peptide stimulating said subgroup of T-cellsto about the same degree as said another peptide.
 3. The pharmaceuticalformulation of claim 1 wherein said peptide consists of the sequenceDENPVVHFFKNIVTPRTPP.
 4. The pharmaceutical formulation of claim 1 saidformulation being a solid oral dosage form.
 5. The pharmaceuticalformulation of claim 1 said formulation being a liquid form adapted forparenteral administration.